1. Field of the Invention
The present invention relates to a method to identify an interface residue of a biomolecular complex, and provides a method for identifying the contact interface in a biomolecular complex accurately. By identifying residues at interface of a complex composed of biomolecules such as proteins, nucleic acids, etc., by the cross-saturation phenomena, the present invention makes it possible to identify the contact interface more accurately as compared to traditional methods.
2. Description of the Background
In living organisms, various molecular interactions take part in physiologic processes and comprise complicated networks. For example, such networks are observed in the immune system with antibodies and T lymphocytes, physiological regulation by hormones, gene expression by transcription factors, signal transmission pathways, etc. Revealing these networks of molecular interactions is an important goal in medicinal research because there are strong possibilities that the results will lead to the development of new medical treatment in the future. Furthermore, to identify various biomolecular interaction sites is expected not only to lead the investigation into physiological processes at molecular level, but also to provide important information for the design of novel drugs targeting the interaction sites (cf. De Vos et al., 1992; Song et al., 1998; Clackson et al., 1995).
One of the methods to identify the contact interface of complex is to determine the tertiary molecular structure using the X-ray crystallographic analysis. However, it is not always easy to use this method for the determination of large protein complex structures. Moreover, this method requires significant amounts of time. In another widely-used method, a combination of replacements of amino acid residues which comprise of the molecular surface and measurements of the binding activity gives only the indirect information about the binding site (cf. Wells et al., 1991; Cunningham et al., 1997).
On the other hand, in regards to NMR methods to identify the residues on the contact interface of biomolecular complex such as proteinxe2x80x94protein complex, protein-nucleic acid complex, etc., the chemical shift peturbation (changes) of backbone amide groups upon the complex formation (cf. Foster et al., 1998) and the changes of the hydrogen-deuterium (H-D) exchange rates (cf. Paterson et al., 1990) have been used as the indicators.
For the purpose of illustration, a schematic drawing of protein A and immunoglobulin G are shown in FIG. 1. Protein A is an immunoglobulin binding protein that is a component of the cell wall of Staphylococcus aureus, and is known to bind specifically with the Fc region of immunoglobulin G (cf. Langone et al., 1982). The extracellular region of protein A is composed of five highly homologous and continuous Fc binding domains, which are called E, D, A, B, and C in order from the N-terminus (end). In addition, there is a cell wall-binding region called X on the C-terminus.
The tertiary structure of the B domain of protein A (FB) in solution has been determined by NMR. The important residues for the binding for the Fc fragment have been identified by the change of the H-D exchange rate and the chemical shift perturbation analyses (cf. Torigoe et al., 1990; Gouda et al., 1992; Gouda et al., 1998).
The three-dimensional structure of FB bound to the Fc fragment has previously been determined by X-ray crystallography (cf. Deisenhofer et al., 1981). In FIG. 2, the interaction interfaces determined by X-ray crystal structure, the H-D exchange experiments, and the chemical shift perturbation are shown. In this figure, CPK models of the FB that indicate the residues on the contact interfaces revealed by X-ray crystallography and the NMR methods are shown. A comparison of these residues indicates that distributions of the residues on the contact interfaces identified by the NMR methods are similar, but not identical, to those revealed by the X-ray crystallography. In particular, the changes in the chemical shifts and the H-D exchange rates induced by binding to the Fc fragment occur in some residues, which contain ones that do not exist on the contact interface revealed by the X-ray crystallography. The reason for the contradiction between the results obtained by the NMR methods and those obtained by the X-ray crystallography may be the result of the fact that changes in the chemical shifts and the H-D exchange rates are also affected by local environment and/or subtle conformational changes induced by the binding to the Fc fragment.
Thus, traditional NMR methods are insufficient for the accurate determination of the contact interface of a biomolecular complex, such as proteinxe2x80x94protein complexes, and an alternative method is needed to identify accurately the contact interfaces.
It is an object of the present invention to provide a method of accurately determining the contact interfaces of biomolecular complexes by identifying the residues present at the contact interfaces.
In order to solve the problems described above, the present inventors, at first, have examined traditional methods in detail to develop a method for a more accurate determination of contact interfaces of biomolecular complexes. So far, for example, high resolution NMR methods have determined the contact interfaces of large proteinxe2x80x94protein complexes (MW greater than 50,000) using the changes in the chemical shifts and the H-D exchange rates induced by formation of the complexes as indicators (cf. Foster et al., 1998; Paterson et al., 1990). However, the interfaces determined by those methods were not always identical to those determined by X-ray crystallography, and there were many ambiguous points. It was found that these methods were limited, and did not always provide accurate analyses. As the result of extensive examination about the identification methods using NMR, it has been revealed that the contact interfaces of large proteinxe2x80x94protein complexes can be determined more accurately by making use of the deuterium labeling and the cross-saturation phenomena. The present invention is based on this finding.
Accordingly, the present invention provides a method to identify interface residues in a biomolecular complex comprising at least two biomolecules, which comprises:
exchanging at least a portion of nonexchangeable hydrogens, i.e., protons and at least 70%, i.e., not less than 70%, of exchangeable hydrogens (protons) in one biomolecule component of the complex, respectively, to deuteriums; and
identifying the position of exchangeable proton(s) which are located on this biomolecule which are present within 10 angstrom (xc3x85) from hydrogen(s) (proton(s)) in a neighboring biomolecule in the complex and receive cross-saturation (are cross-saturated) by cross-saturation phenomena through the interface of the complex.
The present invention makes it possible to identify the contact interface of biomolecular complex more accurately as compared to traditional methods. In a case where the biomolecular complex consists of protein A and protein B, it is possible to determine the conformation (steric configuration) of protein A-protein B complex only on the basis of the conformations (steric configurations) of the each component protein. Furthermore, the minimum structural unit required for binding to protein B can be revealed by the identification of the contact interface of protein A.
Therefore, by mimicking this contact interface, it is possible to prepare a low molecular weight compound which can bind to a protein B. When protein B is a receptor, the low molecular weight compounds may function as an agonist or an antagonist. Thus, the information provided by the inventive method enables the production of new medicines. A particularly preferred embodiment of the present invention is a method to identify interface residues in a biomolecular complex, which comprises:
selecting from among plural biomolecules which compose a complex, one biomolecule with residues to be identified at the complex interface;
exchanging the nonexchangeable hydrogens (protons) and at least 70% (not less than 70%) of exchangeable hydrogens (protons) of the selected biomolecule, respectively, to deuteriums; and
identifying the position of the exchangeable hydrogens (protons) which are located on this biomolecule within 10 angstrom (xc3x85) from hydrogen(s) (proton(s)) in the neighboring biomolecule in the complex and receive cross-saturation (are cross-saturated) by cross-saturation phenomena through the interface of the complex.
In another embodiment, the present invention provides a method for identifying an exchangeable hydrogen atom present at the interface of a biomolecular complex, wherein the biomolecular complex comprises a first biomolecule and a second biomolecule, and wherein at least a portion of the nonexchangeable hydrogens and at least 70% of exchangeable hydrogens in the first biomolecule are exchanged to deuteriums, comprising:
irradiating the second biomolecule in the biomolecular complex; and
identifying at least one exchangeable hydrogen atom in the first biomolecule of the biomolecular complex which receives cross-saturation from the irradiated second biomolecule.